Mitochondrial deoxyguanosine kinase mutations and mitochondrial DNA depletion syndrome

FEBS 27799

FEBS Letters 554 (2003) 319^322

Mitochondrial deoxyguanosine kinase mutations and mitochondrial DNA depletion syndrome Liya Wang , Sta¡an Eriksson Department of Molecular Biosciences, Section of Veterinary Medical Biochemistry, SLU, The Biomedical Centre, P.O. Box 575, SE-751 23 Uppsala, Sweden Received 25 August 2003; revised 3 October 2003; accepted 6 October 2003 First published online 20 October 2003 Edited by Vladimir Skulachev

Abstract Mitochondrial deoxyguanosine kinase (dGK) catalyzes the initial phosphorylation of purine deoxynucleosides. Mutations in the dGK gene leading to de¢ciency in dGK activity is one of the causes of severe mitochondrial DNA depletion diseases. We used site-directed mutagenesis to introduce the clinically observed genetic alterations in the dGK gene and characterized the recombinant enzymes. The R142K enzyme had very low activity with deoxyguanosine and no activity with deoxyadenosine. The E227K mutant enzyme had unchanged Km values for all its substrates but very low Vmax values. C-terminal truncated dGK proteins were inactive. These results may help to de¢ne the role of dGK in mitochondrial DNA (mtDNA) precursor synthesis. - 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Key words: Deoxyguanosine kinase; Mutation; DNA precursor; Mitochondrial DNA depletion

1. Introduction The synthesis of mitochondrial DNA (mtDNA) is not cell cycle regulated and a constant supply of deoxyribonucleoside triphosphate (dNTP) is vital for the maintenance of mitochondrial integrity. However, there is apparently no de novo nucleotide biosynthesis in the mitochondria. Nucleotides required for mtDNA replication are either synthesized by the salvage enzymes in the mitochondria or imported from the cytosol [1,2]. Mitochondrial deoxyguanosine kinase (dGK) and thymidine kinase 2 (TK2) are the two enzymes which are responsible for the initial rate-limiting phosphorylation of all the four deoxynucleosides in the synthesis of precursors for mtDNA replication. Both dGK and TK2 are constitutively expressed in all tissues and the levels of these two enzymes are believed to correlate to the number of mitochondria [1,2]. In resting cells and terminally di¡erentiated cells the de novo synthesis of deoxynucleotides is not present nor is the salvage synthesis of thymidine nucleotides via thymidine kinase 1. In this case the import of nucleotides from the cytosol to the mitochondria is not possible, and the salvage of deoxy-

*Corresponding author. Fax: (46)-18-55 07 62. E-mail address: [email protected] (L. Wang). Abbreviations: dGK, deoxyguanosine kinase; TK2, thymidine kinase 2; dGuo, deoxyguanosine; dAdo, deoxyadenosine; mtDNA, mitochondrial DNA; MDS, mitochondrial DNA depletion syndrome

nucleosides by dGK and TK2 is the only source of DNA precursors. Mitochondrial DNA depletion syndrome (MDS) is a clinically heterogeneous group of disorders characterized by a reduction in mtDNA copy number. Primary mtDNA depletion is inherited as an autosomal recessive trait and may a¡ect single organs, typically muscle or liver, or multiple tissues [3]. Recently, the myopathic form of MDS has been ascribed to mutations in the TK2 gene [4], and the hepatocerebral form has been ascribed to mutations in the dGK gene [5]. Since then, several additional mutations have been identi¢ed in either the TK2 or the dGK genes of patients presenting with MDS [6^10]. In order to understand the mechanism resulting in the disease phenotypes caused by dGK de¢ciency we used site-directed mutagenesis to introduce the mutations, base pair deletions and insertion found in the patients reported recently [5^7]. The recombinant dGK enzymes were expressed in Escherichia coli, puri¢ed and characterized with the most important substrates. 2. Materials and methods 2.1. Materials Radiolabelled nucleosides [8-3 H]-2P-deoxyguanosine (3 H-dGuo; 5.9 Ci/mmol) and [2, 8-3 H]-2P-deoxyadenosine (3 H-dAdo; 17.1 Ci/mmol) were purchased from Moravek biochemicals Inc. Non-radioactive nucleosides were from Sigma. 2.2. Mutagenesis, expression and puri¢cation Point mutations were introduced by a polymerase chain reaction (PCR)-based method as described [11] and the PCR fragments which contained the desired mutations were subcloned into the pET-9d vector (Novagen) as was wild-type dGK cDNA [12]. The mutations were veri¢ed by sequencing using the BigDye1 terminator kit and ABI Prism 310 system (Perkin-Elmer). The plasmids which contain the desired mutations were transformed into the BL21 (DE3) pLysS bacteria (Novagen). All mutant dGK enzymes were expressed and puri¢ed by the same procedure as the wild-type dGK as described previously [13] with the exception that no adenosine triphosphate (ATP) was added in the elution bu¡er. Aliquots of puri¢ed enzymes were analyzed by 12% sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) and protein concentration was determined by Bio-Rad protein assay using bovine serum albumin (BSA) as standard. 2.3. Enzyme assay dGK activity was determined by using [3 H]dGuo or [3 H]dAdo as substrates as previously described [12,14]. The standard reaction mixture contains 50 mM Tris/HCl, pH 7.6, 2 mM ATP, 2 mM MgCl2 , 0.5 mg/ml BSA, 5 mM dithiothreitol and 50 WM labelled deoxynucleoside and puri¢ed enzyme. For the measurement of the kinetic constants a wide concentration range of each substrate was used. The data were

0014-5793 / 03 / $22.00 F 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0014-5793(03)01181-5

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Table 1 Summary of the known genetic defects in the dGK gene Mutations

Outcome (dGK enzyme alteration)

Disease onset and survival

A (nucleotide 204) deletiona C (nucleotide 313) to Tb GT (nucleotides 609^610) deletionc GATT (nucleotides 763^766) duplicationc G (nucleotide 425) to Ac G (nucleotide 679) to Ac

frame shift and premature stop at 80 amino acids premature stop at 105 amino acids premature stop at 213 amino acids premature stop at 255 amino acids

from birth to 6 months, death before 1 year of age From birth, died at 5 months of age from 2 months, died at 6 months of age from 2 months, died at 5 months of age

point mutation R142K point mutation E227K

this patient was compound heterozygous from 6 months, underwent liver transplantation at 17 months; this patient is alive at 5 years of age

a

From [5]. From [7]. c From [6]. b

analyzed by the Sigma Plot Enzyme Kinetic Module version 1.1 (SPSS Inc.).

3. Results 3.1. Genetic alteration in the dGK gene of patients with MDS To date there are ¢ve reported alterations in the dGK gene, including deletions, point mutations or base pair insertion, which caused MDS in patients with hepatic failure as summarized in Table 1. Four of these alterations resulted in premature stop and truncation of the dGK proteins and the a¡ected individuals had severe liver diseases and died at early infancy. The point mutations found in a compound heterozygous patient had milder e¡ects and the patient survived after liver transplantation [5^7]. In order to correlate the disease phenotype to dGK enzyme de¢ciency we used site-directed mutagenesis to introduce these genetic alterations in the dGK gene and expressed the recombinant enzymes in E. coli. They were subsequently puri¢ed and characterized with regard to substrate speci¢city and catalytic e⁄ciency. 3.2. Truncations at the C-terminal end of the dGK proteins A GATT (nucleotides 763^766) duplication in exon 7 resulted in a 22 amino acid deletion at the C-terminus. This C-terminal truncated dGK protein was expressed in E. coli and puri¢ed. The recombinant protein was inactive. To verify the functional importance of the C-terminal domain of dGK, two other C-terminal deletion mutants were generated in the same way with 11 and 18 amino acid truncations. Both recombinant enzymes were inactive. These results suggested that the C-terminal domain is indispensable for enzyme activity. Thus, one can predict that other truncated dGK proteins (Table 1) are most likely inactive.

3.3. Properties of the R142K mutant enzyme Recombinant R142K enzyme had very low activity with dGuo and no detectable activity with dAdo. With dGuo as substrate the Km value was about 30-fold higher and the Vmax value was about 20-fold lower as compared to wild-type dGK (Table 2). Therefore, the e⁄ciency for dGuo phosphorylation by the R142K mutant enzyme was only about 0.2% of the wild-type enzyme. The ATP/MgCl2 dependence was also examined, the Km value was about 11-fold higher and the Vmax value was about 20-fold lower as compared with the wild-type enzyme (Table 3). 3.4. Properties of the E227K mutant enzyme Recombinant E227K enzyme had low activity with both dGuo and dAdo. The Km values for dGuo, dAdo and ATP/ MgCl2 with either dGuo or dAdo as the ¢xed substrate were similar to that of the wild-type dGK (Tables 2 and 3). However, the Vmax values were much lower; 27-, 22-, 31- and 58fold lower respectively, as compared with the wild-type enzyme (Tables 2 and 3). Thus, the e⁄ciencies for dGuo and dAdo phosphorylation were only 2.8 and 5.5% of the wildtype dGK (Table 2). 4. Discussion MDS is characterized as quantitative reduction of mtDNA copy number, which impairs the synthesis of respiratory chain components [3]. The synthesis of mtDNA is carried out by several nuclear encoded proteins, e.g. DNA polymerase Q. However, mtDNA copy number has been suggested to be controlled by the mitochondrial dNTP pools [15]. De¢ciency in either dGK or TK2 activity would impair the synthesis of DNA precursors in the mitochondria and lead to imbalance

Table 2 Kinetic parameters of wild-type and mutant dGK enzymes with phosphate acceptors dGuo a

Wild-type R142K E227K

dAdo

Km (WM)

Vmax (units)

E⁄ciency (Vmax /Km )

Km (WM)

Vmax (units)

E⁄ciency (Vmax /Km )

4 123 L 1 5.2 L 1

43 2.0 L 0.01 1.6 L 0.1

10.8 (100) 0.02 (0.19) 0.3 (2.8)

467 NA 414 L 54

429 NA 19.6 L 1.6

0.9 (100) 0.05 (5.5)

For the measurement of phosphate acceptor constants the dGuo or dAdo concentration was varied (4^505 WM) and the concentration of ATP/ MgCl2 was constant (2 mM). One unit is de¢ned as the formation of 1 nmol dGMP (or dAMP) per min per mg protein. Data in parentheses are relative values as compared with the wild-type enzyme. NA: no detectable activity. a Data from [13].

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Table 3 Kinetic parameters with ATP/MgCl2 for both wild-type and mutant dGK enzymes dGuo (100 WM as acceptor) Wild-type R142K E227K

dAdo (100 WM as acceptor)

Km (WM)

Vmax (units)

E⁄ciency (Vmax /Km )

Km (WM)

Vmax (units)

E⁄ciency (Vmax /Km )

8.5 L 1 97.4 L 1 14 L 2

22 L 0.6 1.1 L 0.01 0.68 L 0.01

2.6 (100) 0.2 (7.7) 0.05 (1.9)

269 L 28 NA 242 L 40

215 L 7 NA 3.7 L 0.2

0.80 (100) 0.02 (2.5)

For the measurement of phosphate donor constant ATP/MgCl2 was varied (20^2000 WM) and dGuo or dAdo was at ¢xed concentration. One unit is de¢ned as the formation of 1 nmol dGMP (or dAMP) per min per mg protein. Data in parentheses are relative values as compared with the wild-type enzyme. NA: no detectable activity.

of dNTP pools, which eventually would cause mtDNA depletion. Human dGK has been characterized enzymatically [12,13,16] and the three-dimensional (3-D) structure is known [17]. The C-terminal K-helix number 9 (K9) of dGK is part of the phosphate donor binding site and therefore plays an important role in enzyme catalysis [17,18]. Deletion of the C-terminal 22 amino acid residues resulted in an incomplete phosphate donor binding site. Indeed the C-terminal 22 amino acid deletion mutant is devoid of enzyme activity, which demonstrated the importance of K9 in enzyme catalysis. Additionally, deletions of part of K9, e.g. 11 and 18 amino acid truncations, resulted in virtually inactive enzymes. Therefore, one may assume that mutations that caused premature stops in the dGK gene would lead to dGK de¢ciency in the tissues of the a¡ected individuals. Two point mutations, i.e. R142K and E227K, have also been identi¢ed in patient with MDS [6]. These mutant enzymes were constructed and characterized. Both residues, R142 and E227, are conserved among the family of deoxynucleoside kinases, including dGK and TK2. R142 is located close to the active site but E227 is not. Residue E227 is located in K8. It forms hydrogen bonds with residues Y191 and Q193 in L4 and appears not to interact directly with any of the substrates [6,17,18]. Experimental results with recombinant E227K enzyme indicated no alteration of substrate binding capacity as revealed by the Km values but it showed a severely decreased catalytic rate. The hydrogen bonds between K8 and L4 probably stabilize the enzyme active site structure. The so-called LID region is a connecting loop between K7 and K8 [17,18] and it is believed that the LID structure undergoes dynamic movements during catalysis. Substitution of glutamate with a lysine residue in this position, E227K, introduced an opposite charge in the area and also a loss of two hydrogen bonds to Y191 and Q193. Such a disturbance may impair enzyme function which is re£ected in low catalytic rates. In contrast to the E227K mutation, the R142K mutation a¡ected the binding of all the substrates; the mutant enzyme had apparently lost its ability to phosphorylate dAdo and also showed very low e⁄ciency for dGuo phosphorylation. In the dGK 3-D structure R142 is situated in the active site, and interacts by a hydrogen bond with the 5P-OH group of the nucleoside [17]. The other amino acid residue that forms a hydrogen bond with the 5P-OH group is E70 and their contacts with the base largely determine the substrate speci¢city (Fig. 1) [17,18]. The amino group of K142 is between 1 and P further away from the 5P-OH group as compared with 2 A R142 (Fig. 1). This clearly diminished the catalytic capacity of dGK, possibly by not being able to stabilize the transition state of the reaction intermediates. The R142K mutant is

thus providing a start point for detailed studies of the reaction mechanism for this class of deoxynucleoside kinases. Translation of mtDNA-encoded proteins is dependent on mtDNA copy number since the expression of mtRNA polymerase is directly correlated to mtDNA copy number [19]. Therefore, mtDNA depletion will result in a reduced amount of mtDNA-encoded enzymes and thus low respiratory activity that, in turn, leads to low ATP content. In the in vivo situation the combination of a poor dGK enzyme and low ATP concentration may result in very low deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate pools as compared with deoxythymidine triphosphate and deoxycytidine triphosphate pools. Liver, heart, skeletal muscle and

Fig. 1. A model of the dGK structure with the active site residues Glu70 and Arg142 or Lys142 that are in close contact with the 5POH group of the substrate (deoxyguanosine: dG). The model is based on the structure of dGK with bound ATP [17], where the adenine base and deoxyribose have been substituted by dG and the mutant Lys142 has been aligned to ¢t the position of Arg142 as close as possible. Color indications: carbon atoms are in black, oxygen atoms are in red, nitrogen atoms are in blue except that in Arg142 the nitrogen atoms are in yellow for comparison.

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brain are among the most energy-dependent tissues of the body and therefore they are vulnerable to mtDNA depletion, most likely through the operation of this vicious circle. The residual dGK activity seems to play a crucial role in disease progression. Patients who completely lacked dGK activity had in common early onset of the disease and died at earlier infancy. The patient who was compound heterozygous with point mutations, e.g. R142K and E227K, had about 27% dGK activity as compared with control individuals [6]. Even though the mutant enzymes had very low activity, the onset of the symptoms was later as compared with other cases and the symptoms were milder. The establishment of a correlation between the dGK activity levels and disease development may encourage development of therapeutic regimes to help patients to suppress the disease. Organ transplantation or gene therapy are among the possible approaches but they may not be available to all affected individuals. Therapies which stimulate nucleotide biosynthesis either via the de novo or the salvage pathway to increase the cellular dNTP pools may increase the chance to delay disease development. In line with this assumption, it was recently reported that partial reversion of mtDNA depletion due to dGK de¢ciency could be obtained by providing resting patient ¢broblast with deoxyadenosine monophosphate (dAMP) or deoxyguanosine monophosphate added to the culture medium [20]. Acknowledgements: This work was supported by grants from the Swedish Medical Research Council and the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning. The authors would like to thank Dr. Hans Eklund for valuable discussions and help with production of the ¢gure.

References [1] Arne¤r, E.S.J. and Eriksson, S. (1995) Pharmacol. Ther. 67, 155^ 186. [2] Eriksson, S. and Wang, L. (1997) Nucleosides Nucleotides 16, 653^659.

[3] Moraes, C., Shanske, S., Tritschler, H., Aprille, J., Andreetta, F., Bonilla, E., Schon, E. and DiMauro, S. (1991) Am. J. Hum. Genet. 48, 492^501. [4] Saada, A., Shaag, A., Mandel, H., Nevo, Y., Eriksson, S. and Elpeleg, O. (2001) Nat. Genet. 29, 342^344. [5] Mandel, H., Szargel, R., Labay, V., Elpeleg, O., Saada, A., Shalata, A., Anbinder, Y., Berkowitz, D., Hartman, C., Barak, M., Eriksson, S. and Cohen, N. (2001) Nat. Genet. 29, 337^341. [6] Salviati, L., Sacconi, S., Mancuso, M., Otaegui, D., Camano, P., Marina, A., Rabinowitz, S., Shi¡man, R., Thompson, K., Wilson, C., Feigenbaum, A., Naini, A., Hirano, M., Bonilla, E., DiMauro, S. and Vu, T. (2002) Ann. Neurol. 52, 311^317. [7] Taanman, J., Kateeb, I., Muntau, A., Jaksch, M., Cohen, N. and Mandel, H. (2002) Ann. Neurol. 52, 237^239. [8] Mancuso, M., Salviati, L., Sacconi, S., Otaegui, D., Camano, P., Marina, A., Bacman, S., Moraes, C., Carlo, J., Garcia, M., Garcia-Alvarez, M., Monzon, L., Naini, A., Hirano, M., Bonilla, E., Taratuto, A., DiMauro, S. and Vu, T. (2002) Neurology 59, 1197^1202. [9] Vila, M., Segovia-Silvestr, T., Gamez, J., Marina, A., Naini, A., Meseguer, A., Lombes, A., Bonilla, E., DiMauro, S., Hirano, M. and Andreu, A. (2003) Neurology 60, 1203^1205. [10] Carrozzo, R., Bornstein, B., Lucioli, S., Campos, Y., de la Pena, P., Petit, N., Dionisi-Vici, C., Vilarinho, L., Rizza, T., Bertini, E., Garesse, R., Santorelli, F. and Arenas, J. (2003) Hum. Mutat. 21, 453^454. [11] Wang, L., Saada, A. and Eriksson, S. (2003) J. Biol. Chem. 278, 6963^6968. [12] Wang, L., Hellman, U. and Eriksson, S. (1996) FEBS lett. 390, 39^43. [13] Herrstro«m Sjo«berg, A., Wang, L. and Eriksson, S. (1998) Mol. Pharmacol. 53, 270^273. [14] Wang, L., Karlsson, A., Arne¤r, E.S.J. and Eriksson, S. (1993) J. Biol. Chem. 268, 22847^22852. [15] Tang, Y., Schon, E., Wilichowski, E., Vazquez-Memije, M., Davidson, E. and King, M. (2000) Mol. Biol. Cell 11, 1471^1485. [16] Herrstro«m Sjo«berg, A., Wang, L. and Eriksson, S. (2001) Antimicrob. Agents Chemother. 45, 739^742. [17] Johansson, K., Ramaswamy, S., Ljungcrantz, C., Knecht, W., Piskur, J., Munch-Petersen, B., Eriksson, S. and Eklund, H. (2001) Nat. Struct. Biol. 8, 616^620. [18] Eriksson, S., Munch-Petersen, B., Johansson, K. and Eklund, H. (2002) Cell. Mol. Life Sci. 59, 1327^1346. [19] Seidel-Rogol, B. and Shadel, G. (2002) Nucleic Acids Res. 30, 1929^1934. [20] Taanman, J.-W., Muddle, J.R. and Muntau, A.C. (2003) Hum. Mol. Genet. 12, 1839^1845.

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